褪黑素对过氧化氢诱导的人晶状体上皮细胞凋亡及自噬的影响

Effect of melatonin on hydrogen peroxide-induced apoptosis and autophagy in human lens epithelial cells

目的 探讨褪黑素对H_(2)O_(2)诱导的人晶状体上皮细胞(LECs)氧化损伤的作用机制。方法 将培养好的人晶状体上皮细胞株(SRA01/04)随机分为对照组(不做任何处理)、H_(2)O_(2)组(400μmol·L^(-1)H_(2)O_(2)处理)、DMSO+H_(2)O_(2)组(400μmol·L^(-1)H_(2)O_(2)和0.2μmol·L^(-1)DSMO处理)、褪黑素+H_(2)O_(2)组(400μmol·L^(-1)H_(2)O_(2)和50μmol·L^(-1)褪黑素处理)。细胞分组处理后继续培养24 h,进行各项实验室检测比较。CCK8实验检测各组细胞活力;免疫印迹实验检测各组细胞凋亡相关蛋白(Bax和Bcl-2)以及自噬相关蛋白(P62、Beclin-1、LC3)的表达水平;TUNEL法检测各组细胞凋亡情况。结果 与DMSO+H_(2)O_(2)组相比,褪黑素+H_(2)O_(2)组SRA01/04细胞活力升高,Bax蛋白表达水平降低(P<0.001), Bcl-2蛋白表达水平增加(P<0.01),SRA01/04细胞凋亡率下降。与DMSO+H_(2)O_(2)组相比,褪黑素+H_(2)O_(2)组SRA01/04细胞中LC3-I、LC3-II及Beclin-1蛋白表达水平均升高(均为P<0.001),P62蛋白表达水平差异无统计学意义(P>0.05),SRA01/04细胞的自噬能力增强。结论 褪黑素能减轻H_(2)O_(2)诱导的LECs细胞凋亡并增强自噬形成,有望为褪黑素在年龄相关性白内障中的作用机制研究提供新的思路。

Objective To explore the effect of melatonin on H_(2)O_(2)-induced oxidative damage in human lens epithelial cells(LECs).Methods The cultured LECs(SRA01/04)were randomly divided into the control group(without any treatment),H_(2)O_(2) group(400μmol·L^(-1) H_(2)O_(2) treatment),DMSO+H_(2)O_(2) group(400μmol·L^(-1) H_(2)O_(2) and 0.2μmol·L^(-1) DMSO treatment),and melatonin+H_(2)O_(2) group(400μmol·L^(-1) H_(2)O_(2) and 50μmol·L^(-1) melatonin treatment).After grouping,cells were cultured again for 24 hours.Cell viability was detected by CCK-8.The expression levels of apoptosis-related proteins,such as B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X(Bax),and autophagy-related proteins,such as P62,Beclin-1 and microtubule-associated protein 1A/1B-light chain 3(LC3),were measured by immunoblotting.Cell apoptosis was detected by TUNEL.Results Compared with the DMSO+H_(2)O_(2) group,the melatonin+H_(2)O_(2) group showed an increase in SRA01/04 cell viability,a decrease in Bax expression level(P<0.001),an increase in Bcl-2 expression level(P<0.01),and a decrease in SRA01/04 cell apoptosis.Compared with the DMSO+H_(2)O_(2) group,the expression levels of LC3-I,LC3-II and Beclin-1 proteins in SRA01/04 cells in the melatonin+H_(2)O_(2) group increased(all P<0.001),while the expression level of P62 showed no significant change(P>0.05),and the autophagy of SRA01/04 cells was enhanced.Conclusion Melatonin attenuates H_(2)O_(2)-induced apoptosis and enhances autophagy in LECs,which is expected to provide new ideas for the study of the effect of melatonin on age-related cataract.